Epigenetic Control of C/EBPa by Distant Synergic Regulatory Elements.

Abstract 1470

Poster Board I-493

The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo.

In this study, we demonstrate synergetic regulation of C/EBPa by two distant cis-elemets located 5' and 3' to the gene and their effect on chromatin architecture.

Previous studies have indicated that as much as 4.8 kb of 5' upstream C/EBPa regulatory sequences were unable to express significant levels of reporter gene activity in transgenic mice. Therefore, we initiated a search for important distal elements in the C/EBPa locus. We have applied a combination of 1) comparative analysis of human and mouse genomic sequences; 2) DNase I hypersensitive studies; 3) chromosome conformation capture (3C); 4) analysis of reporter constructs in stable cells lines; and 5) generation and analysis of transgenic mouse lines. This let us to identify the regulatory role of two distal conserved homology elements located at ∼38 kb 5' of the transcription start site (TSS) of murine C/EBPa (corresponding to ∼45 kb 5' of the TSS of human C/EBPa) and at ∼33 kb 3' to TSS of both murine and human C/EBPa. We show that the constructs lacking both distal elements were unable to express C/EBPa mRNA, while addition of each region resulted in detectable (by Northern blot analysis) expression in transgenic animals.

We have observed a cooperative effect of these two regions on C/EBPa expression, a construct carrying both elements expresses ∼2.5-fold level over constructs carrying either one element alone.

We have investigated the mechanism for the increased expression by these distal elements by using deletion constructs. Our results suggest that lack of these elements results in aberrant gene expression due to proximal promoter DNA hypermethylation and point to a novel mechanism in establishment of critical epigenetic marks.